Diabetes mellitus is a disease resulting in significant morbidity and mortality. The total annual direct and indirect costs of diabetes in the Unites States exceeds $90 billion dollars. Insulin-dependent diabetes mellitus (IDDM), because it occurs in a younger population than non-IDDM, accounts for a disproportionate percentage of these costs. Although the acute manifestations of IDDM can be controlled with daily insulin injections, most patients eventually develop sequelae such as blindness, nephropathy, neuropathy, microangiopathy, and cardiovascular disease. Substantial evidence suggests that meticulous control of glycemia will prevent or minimize these sequelae.
A more physiological method of treating diabetes would be pancreas or islet transplantation. Whole or segmental pancreas transplantation has been performed successfully in man and some preliminary evidence suggests that this technique will prevent the sequelae of diabetes in man. However, pancreas transplantation is not trivial surgery; it poses problems with drainage for exocrine secretions and requires a lifetime of immunosuppressive therapy. On the other hand, islet transplantation has certain theoretical advantages--particularly related to the ease of surgery, the absence of extraneous exocrine tissue, and the cryopreservability of isolated islets. More importantly, islets are more amenable to immunoalteration. Various methods have been developed to prolong allograft survival without continuous immunosuppression in rats and mice. The ability to transplant islets without continuous immunosuppression may eventually prove absolutely necessary in man because many immunosuppressive drugs are somewhat toxic to islets.
Recent improvements in the methods of mass islet isolation and several recent clinical reports suggest that islet transplantation is on the verge of becoming a feasible treatment for IDDM. However, several obstacles exist. First, islets comprise only 2% of the human pancreas; yields from human "islet isolation" procedures are extremely variable and several human donor pancreases are often required to generate sufficient islets for a single transplant. Second, islet allograft rejection has proven difficult to manage using conventional methods and, unfortunately, the majority of islet allografts are quickly lost. Third, there are insufficient human donor pancreases available to treat the vast numbers of type I diabetic patients. Therefore, it seems likely that widespread implementation of islet transplantation would require the development of clinical islet xenotransplantation.
In response to this eventuality, many biomedical corporations are spending millions of dollars developing and patenting "bio-artificial pancreas" technologies (i.e., microencapsulation or macroencapsulation of islet tissue). The underlying concept behind these approaches is that the islet tissue is protected from the immune system by a membrane with pore sizes small enough to prevent immunocytes and antibodies from damaging the graft yet large enough for insulin, oxygen, glucose, and nutrients to pass freely.
During the past few years, several clinical islet transplantation centers have devoted extensive effort to develop experimental islet xenotransplantation models using large animals as donors. Most of these studies have centered on porcine, bovine, canine, or non-human primate islets. However, the pancreata in these species, like the human pancreas, are fibrous and do not readily yield large quantities of intact, viable islet tissue. Moreover, generation of islet preparations from large animal donors is expensive and islet yields are variable.
We have developed a unique animal model for islet xenotransplantation utilizing tilapia, a teleost fish, as islet donors (1). The islet tissue in certain teleost fish, called principal islets or Brockmann bodies (BBs), is anatomically distinct from their pancreatic exocrine tissue and can be easily identified macroscopically. Expensive islet isolation procedures, such as required when procuring islet tissue from mammalian pancreases, are unnecessary. The BBs can be simply harvested with a scalpel and forceps. We have shown that tilapia islets transplanted into diabetic nude mice will produce long-term normoglycemia and a mammalian-like glucose tolerance curve (2).
Teleost fish insulin has been used to maintain human diabetics (1). However, it is likely that the immunogenicity of teleost insulin may prevent clinical application for BB xenotransplantation. On the other hand, the production of transgenic fish whose BBs produce humanized insulin may circumvent this problem. Transgenic fish which produce BBs that physiologically secrete humanized insulin, combined with improvements in bioartifical pancreas technology and encapsulation procedures, would eliminate the need for human pancreatic donors and islet isolation procedures.
Until recently, BBs were harvested manually by microdissection while visualized through a dissecting microscope inside a laminar flow hood (3). Although this was much easier and less expensive than the standard procedure of harvesting islets from rodents, it was a time consuming and tedious task. Although it was easy to harvest sufficient islets to perform xenografts in mice, this method was not well-suited to harvest large volumes of islet tissue as would be required for clinical use or large animal studies. Furthermore, microdissection allows us to collect less than 50% of the islet tissue per donor fish (i.e., those large BBs that are easily visible with the naked eye). Therefore, development of a more efficient method of harvesting BBs would be critical for the future application of fish islets as a donor source for clinical and experimental use. We have recently developed a mass-harvesting method (4).
To date, transgenic fish technology has been used to produce hardier fish that will grow rapidly and will tolerate adverse environments (5-6). Most of these efforts have been directed at insertion of growth hormone transgenes. Another approach has been to insert antifreeze genes from species that tolerate very cold waters (i.e., such as winter flounder) into other species so that they will not only survive, but actually thrive in colder water. This approach permits aquaculture in more northerly regions and allows aquaculture stocks to grow year-round, rather than just during the summer growth season.
No previous transgenic fish studies have been directed at the insulin gene. In fish, insulin is primarily a growth hormone whereas, in mammalians, it is primarily a glucostatic hormone (7). Therefore, it is very likely that altering the expression and/or structure of the fish insulin gene may enhance growth. Consequently, a transgenic fish with altered fish insulin gene expression may demonstrate enhanced growth potential.